The development of methods and kits for performing biological processes within the droplets of “water-in-oil” emulsions have made a tremendous contribution to the development of high throughput analysis technologies, particularly for the high throughput nucleic acid sequencing technologies that employ nucleic acid material amplified within emulsion droplets. It will be appreciated that such emulsions have been successfully employed for a number of uses that include in-vitro transcription/translation, what is referred to as directed evolution, and amplification processes. For example, each aqueous droplet of an emulsion is a micro compartment or microreactor within which the process of interest may be conducted in isolation where the many thousands of the droplets are executing the process in a massively parallel fashion. In the more specific example of nucleic acid amplification, the process can proceed with very high efficiency and without contamination from neighboring droplets. In most applications the type of amplification process performed in aqueous emulsion droplets is the well-known Polymerase Chain Reaction (PCR) method which benefits from the highly efficient heat transfer characteristics of the emulsion as well as the biological compatibility of typical water-in-oil emulsions. In addition, many emulsion embodiments for generating sequencable material are amenable to the inclusion of solid phase substrates such as microspheres (i.e. bead type substrates) upon which the amplification products can be immobilized. This effectively sequesters the amplification products so that when the emulsions droplets are broken to recover the products each species of product can be kept separated from the others and subsequently used as a clonal population.
In general water-in-oil emulsions for use in biological contexts are disrupted or “broken” and the biological material released from the droplets is then purified for subsequent use preferably without destruction or modification of the biological integrity or composition. Traditionally, the water-in-oil emulsions have been broken using a solvent such as isopropanol and the components separated by centrifugation methods. In embodiments that employ the centrifugation method with amplified nucleic acid populations sequestered to beads it is preferable to repeat the centrifugation process several times to remove the oil and surfactants that is followed by rinsing with a buffer solution and further centrifugation the remove the isopropanol.
Traditionally, the methods for generating emulsions, amplification, and recovery of products have been manual and require substantial inputs of time and effort by users of the technology. Therefore, it is the object of the described invention to provide an efficient and automated system for generating emulsion, as well as amplifying and extracting biological elements from the emulsion without causing damage or changing the characteristics of those elements.
A number of references are cited herein, the entire disclosures of which are incorporated herein, in their entirety, by reference for all purposes. Further, none of these references, regardless of how characterized above, is admitted as prior art to the invention of the subject matter claimed herein.